Xylanases, produced by many species of filamentous fungi and bacteria, are a group of enzymes with wide commercial utility. A major application of xylanases is for biobleaching pulp in the production of paper. In addition, xylanases have been used as clarifying agents in juices and wines, as enzymatic agents in the washing of precision devices and semiconductors and they are also used for improving digestibility of poultry and swine feed.
Most xylanases exploited for industrial applications are members of Family 11, showing diversity in their biochemical and biophysical properties. For example, thermostable xylanases have been isolated from bacteria (U.S. Pat. No. 6,667,170), fungi (U.S. Pat. No. 6,635,464), or other extreme thermophiles (Lüthi et al. 1990; Winterhalter et al. 1995; Simpson et al. 1991). Alternatively, xylanase performance has been optimised for various industrial applications via protein engineering (e.g. U.S. Pat. No. 5,759,840; U.S. Pat. No. 5,866,408; U.S. Pat. No. 5,405,769; and Turunen et al., 2001).
Successful implementation of xylanase enzymes in industrial applications requires economical production from a host microbe, which secretes the xylanase into the culture broth during submerged fermentation. This is particularly necessary for the large-scale production of xylanases from thermophiles or extreme thermophiles that are difficult to culture or do not secrete sufficiently high levels of protein. Typically, the host microbe for the production of industrial enzymes is a filamentous fungus such as Trichoderma, Aspergillus or Fusarium, an actinomycete such as Streptomyces or a species of Bacillus bacteria. This means that the genes encoding a target xylanase, whether isolated from a different organism or from protein engineering of a xylanase gene from the host organism, must be cloned into the production host in such a way that the gene is operably linked to the DNA sequences that will facilitate its expression and secretion from the host.
Expression and secretion of exogenous proteins by genetic modification of industrial strains of T. reesei has remained a significant challenge for many years (Conesa et al., 2001). Expression of heterologous proteins in T. reesei elicits an Unfolded Protein Response (UPR; Saloheimo et al., 1999), which results from an accumulation of unfolded or misfolded nascent polypeptides in the lumen of the endoplasmic reticulum (ER). Because of the limited information currently available on the mechanisms regulating folding and secretion of the Family 11 xylanases from T. reesei, several strategies have been implemented to facilitate high-level expression of related exogenous xylanases in T. reesei host strains. These include the use of highly inducible promoters, such as those of the T. reesei cellulase genes, and replacement of the native cellulase genes with xylanase expression constructs containing highly inducible promoters.
Expression of bacterial xylanases from T. reesei may require fusion of the xylanase to a carrier T. reesei polypeptide with an intact domain structure, such as the catalytic core or binding domains of the T. reesei mannanase I or CBH II proteins (Paloheimo, et al., 2003). This strategy, alone or in combination with deletion of one or more cellulase gene(s) from the host T. reesei strain, was disclosed in U.S. Pat. No. 6,635,464 and U.S. Pat. No. 6,667,170 to direct the expression thermophilic Family 11 xylanases from both bacterial (A. flexuosa) and fungal (C. thermophilus) sources. Although the carrier polypeptide certainly increased the production and secretion of the heterologous xylanases disclosed in U.S. Pat. No. 6,635,464 and U.S. Pat. No. 6,667,170 from T. reesei host strains, it is not always desirable to have a carrier polypeptide attached to the xylanase enzyme for industrial applications. In these cases, the carrier polypeptide would need to be removed by proteolysis subsequent to secretion of the fusion protein into the culture broth and prior to its use in the application. However proteolysis adds both time and cost to the overall production of the target xylanase due to cost and incubation time required for the proteolysis step itself as well as potential yield losses of the target xylanase during the proteolytic removal of the carrier polypeptide.
This strategy of using a fusion of a target protein to a carrier protein native to the host cell has also been employed successfully to increase the production and secretion of mammalian chymosin from Aspergillus (Van den Brink et al., WO 02/36752 and WO 03/106484). WO 03/106484 discloses further improvements in the production and secretion of glucoamylase-chymosin fusion proteins from Aspergillus by the introduction of an N-glycosylation motif within the artificial linker polypeptide between the chymosin and glucoamylase fusion partners or within the chymosin peptide sequence. However, there was no demonstration of the benefits of chymosin production from Aspergillus via the introduction of a glycosylation motif in a construct not containing a fusion partner.
Sagt et al. (2000) report improvements in secretion of a target protein from a heterologous eukaryotic host via introduction of an N-glycosylation motif within the target protein. In this report, introduction of an N-glycosylation site into the sequence of a hydrophobic mutant of either a fungal cutinase or of native llama antibody fragments resulted in increased secretion of the target protein from Saccharomyces of Pichia yeast host. However, introduction of the glycosylation site into the native fungal cutinase did not result in any increase in expression from the heterologous yeast hosts.
WO 02/02597 reports the production of the FSH-alpha subunit and glucocerebrosidase polypeptides containing a glycosylation site. The goal of these studies was to improve the stability and expression of these polypeptides. However, the applicability of the method was only demonstrated using the addition of short nucleotide sequences encoding the N-glycosylation motif rather than via direct modification of the primary peptide sequence.
It is an object of the present invention to provide modified xylanases exhibiting improved expression.